Connection (or ligation) of two fragments to make a larger molecule or structure is often achieved with the help of the so-called “click chemistry”. This term is used to describe a set of bimolecular reactions that meet the following criteria: reactions should be wide in scope but selective; produce high yield of the product, proceed with reasonable rate under mild conditions; and tolerate broad range of solvents. Among known click reactions is the reaction of azides with acetylenes. The formation of 1,2,3-triazoles in 1,3-dipolar cycloaddition of azides to triple bond is known, but because the activation energy of acetylene-azide cycloaddition is relatively high (ΔG‡ approximately 26 kcal/mol), the reaction is very slow under ambient conditions.
The utility of the reaction of azides with alkynes was expanded by the discovery of Cu (I) catalysis. 1,3-cycloaddition of azides to terminal acetylenes in the presence of catalytic amounts of cuprous salts is facile at room temperature in organic or aqueous solutions. The copper-catalyzed version of the acetylene-azide cycloaddition (a.k.a. azide click reaction) found a broad range of applications from microelectronics to virus labeling, to drug development. However, the use of cytotoxic Cu (I) catalyst has largely precluded application of this click reaction in living systems.
Catalyst-free 1,3-dipolar cycloaddition of azides to cyclooctynes has made possible a bio-compatible version of the azide click reaction. The triple bond incorporated in an eight-membered ring is apparently already bent into the transition state-resembling geometry, thus reducing the activation barrier.
Besides biocompatibility, another major bottleneck in the application of chemical reporters in living system is the lack of spatial and temporal resolution. Photochemical immobilization of carbohydrates, proteins, DNA fragments, antibodies, and other substrates allows for the formation of patterned or gradient arrays on various surfaces. These techniques are widely used in the development of novel high throughput analytical methods. Due to good compatibility of azide click chemistry with various biological substrates, and the robustness of the triazole linker, it has been employed in surface functionalization including, for example, carbohydrate and protein immobilization. However, this immobilization technique was not amenable to patterned modification of the surface. Although SEM-directed electrochemical reduction of Cu(II) to Cu(I) allows the patterning of fluorescent molecules on a glass slide, this method is of limited in scope and practicality.
New methods for ligating fragments to make a larger molecule or structure are needed in the art.